Cha-Xiang Guan

Calcitonin gene-related peptide promotes the wound healing of human bronchial epithelial cells via PKC and MAPK pathways

Calcitonin gene-related peptide (CGRP) is a 37-amino acid neuropeptide derived from the calcitonin gene. CGRP is widely distributed in the central and peripheral neuronal systems. In the lung, CGRP could modulate dendritic cell function, stimulate proliferation of alveolar epithelial cells and mediate lung injury in mice. In this study, we investigated the effect of CGRP on the wound healing of human bronchial epithelial cells (HBECs) in vitro. The results showed that CGRP accelerated the recovery of wound area of monolayer HBECs in a dose-dependent manner. CGRP inhibited the lipopolysaccharide-induced apoptosis in HBECs. The percentage of S phase and G2/M phase was increased in HBECs after CGRP treatment. CGRP upregulated the expression of Ki67 in a dose-dependent manner. Some pathway inhibitors were used to investigate the signal pathway in which CGRP was involved. We found out that PKC pathway inhibitor (H-7) and MAPK pathway inhibitor (PD98059) could partially attenuate the effect of CGRP, which indicated that CGRP might promote the wound healing of HBECs via PKC and/or MAPK dependent pathway by accelerating migration and proliferation, and inhibiting apoptosis.

Soluble Epoxide Hydrolase Inhibitor Attenuates Lipopolysaccharide-Induced Acute Lung Injury and Improves Survival in Mice

ABSTRACT Acute lung injury (ALI) is characterized by rapid alveolar injury, vascular leakage, lung inflammation, neutrophil accumulation, and induced cytokines production leading to lung edema. The mortality rate of patients suffering from ALI remains high. Epoxyeicosatrienoic acids (EETs) are cytochrome P450-dependent derivatives of polyunsaturated fatty acid with antihypertensive, profibrinolytic, and anti-inflammatory functions. EETs are rapidly hydrated by soluble epoxide hydrolase (sEH) to their less potent diols. The aim of this study was to investigate the role of sEH inhibitor trifluoromethoxyphenyl propionylpiperidin urea (TPPU) and EETs in lipopolysaccharide (LPS)-induced ALI of mice. Our studies revealed that inhibition of sEH with TPPU attenuated the morphological changes in mice, decreased the neutrophil infiltration to the lung, pro-inflammatory cytokine levels (IL-1&bgr; and TNF-&agr;) in serum and bronchoalveolar lavage fluid (BALF), and alveolar capillary leakage (lung wet/dry ratio and total protein concentration in BALF). TPPU improved the survival rate of LPS-induced ALI. In addition, in vitro experiments revealed that both TPPU and EETs (11,12-EET and 14,15-EET) suppressed the expression of IL-1&bgr; and TNF-&agr;, and LDH release in RAW264.7 cells. These results indicate that EETs play a role in dampening LPS-induced acute lung inflammation, and suggest that sEH could be a valuable candidate for the treatment of ALI.

Vasoactive intestinal peptide suppresses macrophage-mediated inflammation by downregulating interleukin-17A expression via PKA- and PKC-dependent pathways.

Interleukin (IL)‐17A is a pro‐inflammatory cytokine that markedly enhances inflammatory responses in the lungs by recruiting neutrophils and interacting with other pro‐inflammatory mediators. Reducing the expression of IL‐17A could attenuate inflammation in the lungs. However, whether VIP exerts its anti‐inflammatory effects by regulating the expression of IL‐17A has remained unclear. Here, we show that there is a remarkable increase of IL‐17A in bronchoalveolar lavage fluid (BALF) and lung tissue of mice with acute lung injury (ALI). Moreover, lipopolysaccharides (LPS) stimulated elevated expression of IL‐17A, which was evident by the enhanced levels of mRNA and protein observed. Furthermore, we also found that VIP inhibited LPS‐mediated IL‐17A expression in a time‐ and dose‐dependent manner in an in vitro model of ALI and that this process might be mediated via the phosphokinase A (PKA) and phosphokinase C (PKC) pathways. Taken together, our results demonstrated that VIP might be an effective protector during ALI by suppressing IL‐17A expression.

Aucubin Alleviates Bleomycin-Induced Pulmonary Fibrosis in a Mouse Model

Pulmonary fibrosis is a life-threatening disease characterized by progressive dyspnea and worsening of pulmonary function. No effective therapeutic strategy for pulmonary fibrosis has been established. Aucubin is a natural constituent with a monoterpene cyclic ring system. The potency of aucubin in protecting cellular components against inflammation, oxidative stress, and proliferation effects is well documented. In this study, we investigated the protective effect of aucubin against pulmonary fibrosis in mice. A mouse model of pulmonary fibrosis was established by intratracheal injection of bleomycin (BLM), and aucubin was administered for 21xa0days after BLM injection. We found that aucubin decreased the breathing frequency and increased the lung dynamic compliance of BLM-stimulated mice detected by Buxco pulmonary function testing system. Histological examination showed that aucubin alleviated BLM-induced lung parenchymal fibrotic changes. Aucubin also reduced the intrapulmonary collagen disposition and inflammatory injury induced by BLM. In addition, aucubin reduced the expression of pro-fibrotic protein transforming growth factor (TGF)-β1 and α-smooth muscle actin (α-SMA) of pulmonary fibrosis mice induced by BLM. Furthermore, the effect of aucubin on the proliferation and differentiation of fibroblast was investigated in vitro. Aucubin inhibited the mRNA and protein expression of Ki67 and proliferating cell nuclear antigen (PCNA) induced by TGF-β1 and reduced the cell proliferation in a murine fibroblast cell NIH3T3. Aucubin also reduced the collagen syntheses and α-SMA expression induced by TGF-β1 in fibroblast. Our results indicate that aucubin inhibits inflammation, fibroblast proliferation, and differentiation, exerting protective effects against BLM-induced pulmonary fibrosis in a mouse model. This study provides an evidence that aucubin may be a novel drug for pulmonary fibrosis.

l‐Arginine promotes DNA repair in cultured bronchial epithelial cells exposed to ozone: involvement of the ATM pathway

Ozone may lead to DNA breaks in airway epithelial cells. p‐ATM (phosphorylated ataxia telangiectasia mutated) plays a pivotal role in DNA repair. Derivatives of NO (nitric oxide) are regulators of the phosphorylation, and NO is increased under oxidative stress. The present study was aimed to study the effect of NO donor l‐arg (l‐arginine) on DNA damage repair in human bronchial epithelial cells exposed to ozone and the potential mechanisms involved. HBECs (human bronchial epithelial cells) were cultured with or without ozone (1.5 ppm, 30 min), DNA breaks were measured with a comet assay and agarose gel electrophoresis, cell cycling was determined by flow cytometry and p‐ATM was measured by immunofluorescence and Western blot. Data were analysed by ANOVA (analysis of variance). P<0.05 was considered as significant. Ozone induced marked DNA breaks, G1‐phase arrest and increased expression of p‐ATM in HBECs, while wortmannin reduced the levels of p‐ATM induced by ozone; the NO donor, l‐arg, minimized the effects of ozone‐induced DNA breaks and increased the level of p‐ATM, while the NO synthase inhibitor, l‐NMMA [N(G)‐minomethyl‐l‐arginine], restrained those effects of l‐arg. The effect of l‐arg on DNA repair is NO‐mediated, and p‐ATM is implicated in the processes of DNA repair.

Inhibition of lipopolysaccharide-mediated rat alveolar macrophage activation in vitro by antiflammin-1

Antiflammin‐1 (AF‐1) is a synthetic nonapeptide with a similar sequence to the conserved sequence of CC10 secreted by lung Clara cells. Studies suggest that it is potent inhibitor of inflammation. We investigated the effects and possible mechanisms of AF‐1 on LPS‐induced alveolar macrophage (AM) activation in vitro. AMs harvested from the BALF of Sprague‐Dawley (SD) rat were treated with various concentrations of AF‐1 both simultaneously and after LPS stimulation. The concentrations of the cytokines IL‐1β, IL‐6, and IL‐10 in the supernatant were detected by an enzyme‐linked immunosorbent assay. The mRNA expression levels of these cytokines in AMs were analyzed using quantitative RT‐PCR. To investigate more fully the possible mechanisms by which AF‐1 modulates the expression of cytokines, cells were pretreated with anti‐IL‐10 antibody. Toll‐like receptor‐4 (TLR‐4) expression on the cell surface was also detected using flow cytometry. The results showed that AF‐1 suppressed mRNA expression and protein production of IL‐1β and IL‐6, while it promoted IL‐10 expression in LPS‐stimulated AMs, in a dose‐dependent manner. The inhibitory effects of AF‐1 on IL‐1β were significantly decreased when endogenous production of IL‐10 was blocked. AF‐1 also showed an effect on downregulated TLR‐4 expression in LPS‐stimulated AMs. The data show for the first time that AF‐1 modulates the AM response to LPS by regulating TLR‐4 expression and upregulating IL‐10 secretion, which could be another important mechanism in the AF‐1 inhibiting effect on inflammation.

Vasoactive intestinal peptide overexpression mediated by lentivirus attenuates lipopolysaccharide-induced acute lung injury in mice by inhibiting inflammation

HIGHLIGHTSWe firstly found intratracheal injection of Lenti‐VIP for 7days significantly increased intrapulmonary VIP content in mice.VIP alleviates ALI induced by LPS in vivo.VIP inhibits pro‐inflammatory TNF‐&agr; expression in murine macrophages stimulated by LPS via PKC and PKA pathways. ABSTRACT Vasoactive intestinal peptide (VIP) is one of the most abundant neuropeptides in the lungs with various biological characters. We have reported that VIP inhibited the expressions of TREM‐1 and IL‐17A, which are involved in the initiation and amplification of inflammation in acute lung injury (ALI). However, the overall effect of VIP on ALI remains unknown. The aim of this study is to investigate the therapeutic effect of VIP mediated by lentivirus (Lenti‐VIP) on lipopolysaccharide (LPS)‐induced murine ALI. We found that the expression of intrapulmonary VIP peaked at day7 after the intratracheal injection of Lenti‐VIP. Lenti‐VIP increased the respiratory rate, lung compliance, and tidal volume, while decreased airway resistance in ALI mice, detected by Buxco system. Lenti‐VIP significantly reduced inflammatory cell infiltration and maintained the integrity of the alveolar septa. Lenti‐VIP also remarkably decreased the total protein level, the number of neutrophil and lactate dehydrogenase activity in the bronchoalveolar lavage fluid of LPS‐induced ALI mice. In addition, Lenti‐VIP down‐regulated pro‐inflammatory tumor necrosis factor (TNF)‐&agr; mRNA and protein expression, while up‐regulated anti‐inflammatory interleukin‐10 mRNA and protein expression in lungs of ALI mice. Furthermore, we observed that VIP reduced the TNF‐&agr; expression in murine macrophages under LPS stimulation through protein kinase C and protein kinase A pathways. Together, our findings show that in vivo administration of lentivirus expressing VIP exerts a potent therapeutic effect on LPS‐induced ALI in mice via inhibiting inflammation.

Inhibition of glycolysis alleviates lipopolysaccharide-induced acute lung injury in a mouse model: ZHONG et al.

Gluconic metabolic reprogramming, immune response, and inflammation are intimately linked. Glycolysis involves in the pathologic progress in acute and chronic inflammatory diseases. However, the involvement of glycolysis in the acute lung injury (ALI) is still unclear. This study investigated the role of glycolysis in an animal model of ALI. First, we found that lactate content in serum was remarkably increased in ALI patients and a murine model induced by intratracheal administration of lipopolysaccharide (LPS). The key proteins involving in glycolysis were robustly elevated, including HK2, PKM2, and HIF-1α. Intriguingly, inhibition of glycolysis by 2-deoxyglucose (2-DG) pronouncedly attenuated the lung tissue pathological injury, accumulation of neutrophil, oxidative stress, expression of proinflammatory factors in the lung of ALI mice induced by LPS. The 2-DG treatment also strongly suppressed the activation of the NOD-like receptor (NLR) family and pyrin domain-containing protein 3 (NLRP3) inflammasome. Furthermore, we investigated the role of glycolysis in the inflammatory response of primary murine macrophages activated by LPS in vitro. We found that the 2-DG treatment remarkably reduced the expression of proinflammatory factors induced by LPS, including tumor necrosis factor-α messenger RNA (mRNA), pro-interleukin (IL)-1β mRNA, pro-IL-18 mRNA, NLRP3 mRNA, caspase-1 mRNA, and IL-1β protein. Altogether, these data provide a novel link between gluconic metabolism reprogramming and uncontrolled inflammatory response in ALI. This study suggests glycolytic inhibition as an effective anti-inflammatory strategy in treating ALI.

Effect of EETs on proliferation and apoptosis of lipopolysaccharide-treatment HBECs#

(1 mg/kg/day) was gavage dosing for 21 days. Then the mice were sacrificed. The lift lung lobes were fixed for HE and Masson staining, and the right lobes were used to observe the procollagen I and III expressions by RT-PCR. Results: Histological examination showed that TPPU treatment alleviated the bleomycin-induced inflammation, maintained the alveolar structure. Masson staining indicated that TPPU could attenuate the expression of bleomycin-induced collagen. Furthermore, the expression of procollagen I and III mRNAs were significantly reduced with TPPU treatment. Discussion: TPPU could increase the concentration of EETs in lung, which alleviated the bleomycin-induced inflammatory and collagen deposition, and inhibited the PF in mice. This work was supported by The Innovation Fund for Institution of Higher Education of Hunan Province (11K077), Hunan Provincial Innovation Foundation for postgraduate (CX2011B048), and the Open-End Fund for the Valuable and Precision Instrument of Central South University (CSUZC2012003).